In the developing brain, neurons make connections with their target cells by sending axons tipped with motile growth cones into the cellular environment. These growth cones guide the axon to appropriate targets by responding to molecular cues. Development of axon branches is an important aspect of axon guidance in the mammalian CMS. Calcium signaling in the growth cone is important for determining rates of axon outgrowth and in responding to guidance cues. Growth cones at the tips of branches are exposed to environmental guidance cues different from those that the primary axon senses. Importently, in vivo and in culture axons and their branches exhibit different rates of outgrowth. The work proposed in Specific Aim 1 will investigate the role of localized calcium signaling in regulating the rates of outgrowth of primary axons versus their collateral branches. Using the developing rodent cortex as a model system, I will carry out live cell fluorescence calcium imaging of dissociated cortical neurons in culture. This approach permits precise imaging of calcium dynamics in living neurons. Direct local application of guidance factors and photorelease of caged signaling molecules in different branches of the same axon will enable precise examination of their effects on calcium dynamics in the primary axon ans its branches. In Specific Aim 2, the use of a cortical slice preparation and two-photon fluorescence microscopy will extend this work to a more in vivo model to further investigate the role of calcium signaling in regulating axon growth and branching in the developing mammalian cortex. The ultimate goal of this research is to study how axons grow and branch into their appropriate targets in the developing brain. These developmental processes are important in understanding how the mammalian brain responds to injury, and how it may be induced to regenerate.